Anais da Academia Brasileira de Ciências (2015) 87(1): 109-119 (Annals of the Brazilian Academy of Sciences) Printed version ISSN 0001-3765 / Online version ISSN 1678-2690 http://dx.doi.org/10.1590/0001-3765201520130519 www.scielo.br/aabc

Detection of proteases from Sporosarcina aquimarina and Algoriphagus antarcticus isolated from Antarctic soil

ANDERSON F. SANTOS1,2, FABIANO PIRES1, HUGO E. JESUS1, ANDRÉ L.S. SANTOS1,2, RAQUEL PEIXOTO1, ALEXANDRE S. ROSADO1, CLAUDIA M. D’AVILA-LEVY3 and MARTA H. BRANQUINHA1

1Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, 21941-902 Rio de Janeiro, RJ, Brasil 2Programa de Pós-Graduação em Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Av. Athos da Silveira Ramos, 149, 21941-909 Rio de Janeiro, RJ, Brasil 3Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Laboratório de Biologia Molecular e Doenças Endêmicas, Av. Brasil, 4365, 21040-360 Rio de Janeiro, RJ, Brasil

Manuscript received on December 17, 2013; accepted for publication on April 3, 2014

ABSTRACT Two psychrophilic bacterial samples were isolated from King George Island soil, in Antarctica. The phylogenetic analysis based on the 16S rRNA (rrs) gene led to the correlation with the closest related isolates as Sporosarcina aquimarina (99%) and Algoriphagus antarcticus (99%), with query coverage of 99% and 98%, respectively. The spent culture media from both isolates displayed proteolytic activities detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis containing gelatin as protein substrate. Under the employed conditions, S. aquimarina showed a 55 kDa protease with the best activity detected at pH 7.0 and at 27°C. A. antarcticus also showed a single extracellular protease, however its molecular mass was around 90kDa and its best activity was detected at pH 9.0 and at 37°C. The proteases from both isolates were inhibited by 1,10-phenanthroline and EDTA, two metalloprotease inhibitors. This is the first record of protease detection in both species, and our results may contribute to broaden the basic knowledge of proteases from the Antarctica environment and may help prospecting future biotechnological applications of these enzymes. Key words: Algoriphagus antarcticus, Antarctica, Sporosarcina aquimarina, Protease.

INTRODUCTION surface (Feller and Gerday 2003). In this context, The Earth has a considerable proportion of Antarctica is the coldest, driest, highest, windiest extremely cold environments, represented by the and most isolated and unknown continent on Earth Antarctic and Artic regions, as well as cold regions, (Olson 2002). The microorganisms that survive including the oceans (usually 4–5°C below a depth under these inhospitable conditions play a major of 1,000m), which cover around ¾ of the Earth’s role in the Polar ecosystem, as they are the major components in the Antarctic ecosystem biomass Correspondence to: Marta Helena Branquinha E-mail: [email protected] (Wynn-Williams 1996, Pointing et al. 2009).

An Acad Bras Cienc (2015) 87 (1) 110 ANDERSON F. SANTOS et al.

Due to the low complexity of the food chain and applications include their use in the detergent industry, the low number of individuals and diversity, any textile industry, food industry, bioremediation, and change in the physicochemical conditions of this biocatalysts in low water conditions (Gerday et environment is quickly noticeable in the various al. 2000). Further knowledge of the mechanisms trophic levels (Yergeau and Kowalchuk 2008). involved in the molecular adaptation of enzymes to The great selective pressure exerted by the low temperatures is of great interest to biotechnology. Antarctic environmental conditions determines either In view of this, the scope of this work was to identify the survival, the death or the efficient adaptation two bacteria isolated from King George Island soil, of the organisms, such as that of extremophile in Antarctica, and characterize the extracellular microorganisms known as psychrophiles (Morita proteases produced by both isolates, in order to 1975). Adaptations developed in the cell constituents assess their possible biotechnological potential. include changes that are only found in microorganisms from this region of our planet, such as changes MATERIALS AND METHODS in the membrane structure, energy-generating ISOLATION AND IDENTIFICATION OF BACTERIAL ISOLATES systems, protein synthesis machinery, biodegradable enzymes, components responsible for the nutrient The bacterial isolates in this study were grown incorporation and in the structure of enzymes’ active up from the soil samples collected from different sites, among others (Gerday et al. 1997, 2000, Russel locations at Admiralty Bay, located in the center of 1998, Cavicchioli and Thomas 2000). King George Island in the Southern Shetland Islands, Psychrophilic enzymes are characterized by which lies 130 km northwest off the Antarctic high catalytic efficiency in low temperatures, high Peninsula between latitudes 61°00’S and 63°30’S and level of thermolability and an increased structural longitudes 53°55’W and 62°50’ (Teixeira et al. 2010) flexibility in order to provide better access to (Fig. 1). The collection took place in the 2006/2007 the substrate; in this sense, the protein flexibility austral summer and it received logistic support plays a critical role bestowing adaptability to cold from the Brazilian Navy. Bacterial enumeration and (Thomas and Dieckmann 2002). Despite their isolation were performed in the Brazilian Antarctic usefulness in industrial and domestic processes Station laboratory (Comandante Ferraz). After (Feller and Gerday 2003), psychrophiles and their homogenization of the soil sample in plastic bags, an products are underutilized in biotechnology due aliquot of 5 g was transferred to Erlenmeyer flasks to the thermolability of their enzymes and the cost containing 45 ml of sterile saline solution (0.85% of production and processing at low temperatures NaCl) and manually agitated during 10 min. The (Margesin and Feller 2010). However, advances in soil suspensions were then serially diluted (1:10) in the biotechnology enzymatic area, especially with conical tubes containing 9 ml of sterile saline solution psychrophilic enzymes, have led to a rapid growth in before plating in duplicate on Petri dishes containing this field (Cavicchioli et al. 2002), hence the study of Luria-Bertani (LB) solid medium. The counting was proteolytic enzymes from Antarctica is an interesting performed after four weeks of incubation at 4°C. theme for biotechnology (Feller and Gerday 2003). Pure isolates were transferred to 15-ml conical tubes The world market for industrial enzymes is containing 10ml of slanted Luria Bertani (LB) agar a 2-billion-dollar per year business. Proteases are medium (Bertani 1951) and transported to Brazil among the most commercialized enzymes in the at 4°C. Both the isolates used in this study, named world and account for over 65% of the world’s initially Samples 1 and 2, were maintained in Luria- enzyme market (Rao et al. 1998). Some of their Bertani (LB) solid medium.

An Acad Bras Cienc (2015) 87 (1) PROTEASES FROM S. aquimarina AND A. antarcticus 111

Fig. 1 - Location of collection of bacterial samples: (A) The Antarctic continent, with the Antarctic Peninsula highlighted. (B) Antarctic Peninsula and archipelago of the South Shetland Islands. (C) The Admiralty Bay highlighted in King George Island. The circle indicates the location of the Comandante Ferraz Antarctic Station (EACF), from Brazil. Adapted from Simões et al. (2004).

For the identification of the isolates, the 27f (5’-AGAGTTTGACATGGCTCAG-3’) and primers used in the analysis were 27f (5’- 1492R (5’- GTTTACCTTGTTACGACTT- 3’) AGAGTTTGATCCTGGCTCAG-3’) and 1492R (Dojka et al. 2000), and the intermediate primers (5’-AAGGAGGTGATCCAGCGCA- 3’) due to 532F (5’-CGTGCCAGCAGCCGCGGTAA-3’) the fact that both flank the coding region for the and 907R (5’- CCGTCAATTCMTTTGAGTTT-3’) 16S rRNA (rrs) gene (Massol-Deya et al. 1995). (Ishii and Fukui 2001). The quality of the sequences The amplicons generated by PCR were purified was checked using the Phred program (Ewing with the PCR Purification System (Qiagem) kit for and Green 1998). The sequences obtained were further automatic sequencing in the MEGABACE analyzed in the BLASTn program (http://www. DNA Analysis System 500 (GE Healthcare). The ncbi.nlm.nih.gov/) and Genbank to align them to sequencing was performed using the external primers the sequences stored in the database.

An Acad Bras Cienc (2015) 87 (1) 112 ANDERSON F. SANTOS et al.

PHYLOGENY proteases was calculated by comparison with the For the assembly of the phylogenetic tree, we used mobility of molecular mass standards (Fermentas). the DNA sequences coding for the 16S rRNA of The gels were dried, scanned and the densitometric the two isolates studied, their respective species analysis was digitally processed using Image J obtained in the reference database GenBank and public domain software. other bacterium species of different phylum. All ENZYME CLASS DETERMINATION sequences were converted into FASTA format using BioEdit. MEGA 4.0 software was used to In order to determine the protease class, the gels were assemble the tree. The trees were generated by the incubated for 24 h at pH and temperature conditions neighbor-joining method (Gascuel and Steel 2006), that better resolved the proteolytic profile containing while the model used was Jukes Cantor (Chor et al. the following proteolytic inhibitors: 10 mM phe­ 2006), bootstrap with 10,000 repeats. nylmethylsulfonyl fluoride (PMSF), 100 μM tosyl- L-lysine chloromethyl ketone (TLCK) (serine GROWTH CONDITIONS AND PREPARATION OF protease inhibitors), 10 mM 1,10-phenanthroline, EXTRACELLULAR EXTRACTS 10 mM ethylenediaminetetraacetic acid (EDTA) Bacterial isolates were grown in LB medium for (metalloprotease inhibitors), 10 μM L-trans- 48 h at 4°C. Cultures were passed over a 0.22-µm epoxysuccinyl-leucylamide-(4-guanidino)-butane filtration unit (Millipore), and each spent culture (E-64) (cysteine protease inhibitor) and 1 μM medium was then concentrated 10 times using an pepstatin A (aspartic protease inhibitor). The results Amicon system (Stirred Cell Model 8200) at 4°C were compared with a control group that had been (cut-off 10,000 MW). The protein concentration incubated in the absence of the proteolytic inhibitors. was determined according to Lowry et al. (1951), RESULTS using bovine serum albumin as standard. The results obtained in the sequencing of Samples EXTRACELLULAR PROTEASE DETECTION 1 and 2 showed 99% similarity with the isolate The extracellular proteolytic activity (50 µg protein/ Sporosarcina aquimarina – AF202056 (query slot) was determined using 10% SDS-PAGE with coverage of 99%) and Algoriphagus antarcticus 0.1% copolymerized gelatin as substrate (Heussen – AJ577141 (query coverage of 98%). The and Dowdle 1980). Electrophoresis was performed phylogenetic tree confirmed the grouping of the at 120V, 4°C for 90 min, and the gels were then isolates with the reference species obtained in incubated in 2.5% Triton X-100 for 1 h, washed in GenBank, in addition to revealing the evolutionary distilled water and incubated for 24 h in one of the distance between these two genera with sequences following pH buffers: 10 mM sodium citrate (pH from other bacterial phyla (Fig. 2). 3.0), 50 mM sodium phosphate (pH 5.0 and pH 7.0) The assembly of the phylogenetic tree for and 20 mM glycine-NaOH (pH 9.0 and pH 11.0). both samples was followed by the detection of the The effect of the temperature upon the enzyme extracellular protease activity from S. aquimarina activity was assessed by incubating the gels at 4°C, and A. antarcticus by gelatin-SDS-PAGE. 27°C, 37°C and 56°C. The detection of proteolytic The spent culture media of both species tested bands was performed by staining the gels for 2 h with displayed a single protease band each, representing 0.2% Coomassie Brilliant Blue R-250 in methanol- distinct proteolytic activities. Under the employed acetic acid-water (50:10:40, v/v/v) and destaining experimental setting, the best conditions for in the same solvent. The molecular mass of the the hydrolytic activity detected at 55 kDa in S.

An Acad Bras Cienc (2015) 87 (1) PROTEASES FROM S. aquimarina AND A. antarcticus 113

Fig. 2 - Phylogenetic tree of 16S rRNA gene sequences (around 1500 bp) showing the relationship between two Antarctic bacterial isolates (Samples 1 and 2) and related species identified using BLASTn searches. The tree was constructed based on a neighbor- joining method. Bootstrap analyses were performed with 10,000 repetitions, and results are represented at the branch points. The GenBank accession number of each species is cited in parentheses (S. aquimarina JX45902 and A. antarcticus JX45901). Type species are referred to as (T). aquimarina were found to be at pH 7.0 and at 27 °C the single protease detected at 90 kDa yielded a (Fig. 3 and 4). Nevertheless, the proteolytic band distinct profile, in which the best conditions were was consistently observed at pH values ranging pH 9.0 and 37°C (Fig. 3 and 4). Hydrolysis was from 5.0 to 9.0, displaying approximately 80% of also observed at pH 7.0 (more than 80% of the its total activity at pH 5.0 and more than 60% at total activity) and at pH 5.0 (approximately 20% pH 9.0, as well as at 37°C (approximately 80% of of the total activity) as well as in the temperature the maximum activity detected at 27°C). A drastic range 27-56°C (approximately 40% and 10%, reduction (more than 80%) of the activity was respectively, of the activity detected at 37°C). No observed at pH values 3.0 and 11.0 as well as at proteolytic activity was detected at pH 3.0 and at 4°C and at 56°C. For A. antarcticus, in contrast, pH 11.0 nor at 4°C (Fig. 3 and 4).

An Acad Bras Cienc (2015) 87 (1) 114 ANDERSON F. SANTOS et al.

Fig. 3 - Gelatin-SDS-PAGE showing the detection of extracellular proteases by Sporosarcina aquimarina (a) and Algoriphagus antarcticus (b) at distinct pH values and optimal temperature conditions, as described in Materials and Methods. Molecular masses of the proteases, expressed in kiloDaltons, are represented on the left. The densitometric analysis of the proteolytic halos, expressed as the percentage of the remaining activity with respect to the optimal pH conditions, is shown on the right.

The proteolytic zymograms indicated that the TLCK induced some conformational change in proteases secreted by S. aquimarina and A. antarcticus the enzyme structure so that the active site could were both sensitive to 1,10-phenanthroline and better perform gelatin hydrolysis. EDTA (Fig. 5) at temperature and pH conditions DISCUSSION that better resolved the proteolytic profile, which indicates the presence of metalloproteases in The diversity of microorganisms inhabiting cold the culture supernatant from both isolates. In environments has been extensively investigated over addition, incubation of gels with cysteine protease the past few years with a focus on culture-independent and aspartic protease inhibitors, such as E-64 techniques, but the information obtained by such and pepstatin A, did not alter the proteolytic methods may be complemented by the cultivation of activity in either of the samples (data not such microorganisms (Casanueva et al. 2010). In this shown). Interestingly, the proteolytic activity was sense, Gratia et al. (2009) demonstrated that, after the markedly enhanced in the presence of the serine initial screening to ascertain exoenzyme production protease inhibitor PMSF, which affected the (proteases, lipases, amylases, cellulases and activity in both species, while the serine protease xylanases) in more than 1000 isolates collected from inhibitor TLCK led to higher levels of activity in several cold environments (Antarctica, Kerguelen S. aquimarina (Fig. 5). It is likely that PMSF and Island, Spitzberg, Siberia, Canada, Lapland and other

An Acad Bras Cienc (2015) 87 (1) PROTEASES FROM S. aquimarina AND A. antarcticus 115

Fig. 4 - Gelatin-SDS-PAGE showing the detection of extracellular proteases in Sporosarcina aquimarina (a) and Algoriphagus antarcticus (b) at distinct temperature values and optimal pH conditions, as described in Materials and Methods. Molecular masses of the proteases, expressed in kiloDaltons, are represented on the left. The densitometric analysis of the proteolytic halos, expressed as the percentage of the remaining activity with respect to the optimal temperature conditions, is shown on the right. deep seawater samples), over 100 isolates showed our analysis were properly identified. Despite the the ability to produce at least two of the above- importance of the psychrophilic proteases study, mentioned exoenzymes at 4°C. Taken together, these our work is the first to describe proteases from S. observations clearly indicate that the identification aquimarina, which was originally isolated from of extremophiles and their correlation with enzyme seawater in Korea (Yoon et al. 2001), as well as production may be linked to organismal function from A. antarcticus, originally isolated from and provide a basis for further physiological analysis microbial mats of lakes Reid, Fryxell and Ace in (Casanueva et al. 2010). Antarctica (Van Trappen et al. 2004). Through the phylogenetic analysis performed In order to detect proteolytic activities in these in this work, the studied isolates were more samples, gelatin-SDS-PAGE was employed. Among correlated to S. aquimarina and A. antarcticus, with the many benefits displayed by this technique, it is a 99% similarity. According to Stackebrandt and simple standard method to assess the complexity of Ebers (2006), only similarity values below 98.7- proteases in cell systems, and intensity of bands can 99% are mandatory to require a deeper evaluation, be quantified with densitometric analysis. Although such as DNA-DNA hybridization, and as such we one cannot accurately estimate the molecular mass may conclude that both the samples employed in in such conditions, because of the non-reduction

An Acad Bras Cienc (2015) 87 (1) 116 ANDERSON F. SANTOS et al.

Fig. 5 - Gelatin-SDS-PAGE showing the detection of extracellular proteases in Sporosarcina aquimarina (a) and Algoriphagus antarcticus (b) at pH and temperature conditions that better resolved the proteolytic profile, as described in Materials and Methods, in the absence (Control) or in the presence of different proteolytic inhibitors: 10 mM PMSF, 100 µM TLCK, 10 mM 1,10-phenanthroline (PHEN) and 10 mM EDTA. Molecular masses of the proteases, expressed in kiloDaltons, are represented on the left. The densitometric analysis of the proteolytic halos, expressed as the percentage of the remaining activity with respect to the control, is shown on the right. conditions of the samples, a side-by-side comparison proteases of psychrotolerant bacteria isolated of the protease profiles in distinct physical conditions, from Antarctica have already been previously such as pH and temperature, as well as in the presence classified as metalloproteases, with optimal activity of inhibitors that specifically block the enzymatic at 40°C and pH 7-9 for Pseudomonas sp. strains activity of proteases, provides information about (Vazquez et al. 2004) and at 45°C and pH 6-10 for the occurrence and the expression of these activities Pseudoalteromonas sp. strain P96-47 (Vazquez within a sample group (d’Avila-Levy et al. 2012). et al. 2008). Proteases produced by this class of In our study, the proteolytic zymograms microorganisms apparently are not totally adapted showed the presence of metalloproteases as the only to function in cold environments, even when the extracellular proteolytic activity detected in both original environment from which these bacteria isolates, S. aquimarina and A. antarcticus, with the were isolated hardly reached over 10°C. Although best temperature conditions displayed at 27°C and no activity is displayed in the 0-4°C temperature 37°C, respectively. Concerning the best pH value range, i.e. similar to those of the collection sites, tested, S. aquimarina extracellular metalloprotease we cannot rule out the improvement in the enzymes presented the highest activity at pH 5.0-7.0, performance in their natural environment, which and A. antarcticus at pH 7.0-9.0. Extracellular could be better than the results displayed in vitro

An Acad Bras Cienc (2015) 87 (1) PROTEASES FROM S. aquimarina AND A. antarcticus 117

(Vazquez et al. 2008). This behavior may be due and A. antarcticus species have turned out to be to innumerable cofactors that influence the enzyme promising sources of proteases, which highlights activity present in those environments, and that the need for further studies in order to assess the have not been mimicked in vitro yet. One of the biotechnological potential of these enzymes. most common methods of freeze-protection in cells ACKNOWLEDGMENTS is the accumulation of compatible solutes, which results in a reduction of the freezing point of the This study received financial and logistic cytoplasmic aqueous phase and might also directly support from the Programa Antártico Brasileiro stabilize macromolecules, particularly enzymes. (PROANTAR) as part of the IPY Activity # 403 There are also more specific mechanisms for “MIDIAPI Microbial Diversity of Terrestrial and freeze-protection, such as ice-nucleating proteins Maritime ecosystems in Antarctic Peninsula” and anti-freeze proteins (Casanueva et al. 2010). (520194/2006-3) and financial support from Microorganisms not yet isolated, such as Coordenação de Aperfeiçoamento de Pessoal de protease producers and isolates from the Antarctic Nível Superior (CAPES), Fundação Carlos Chagas environment, may reveal new information Filho de Amparo à Pesquisa do Estado do Rio concerning the enzymes produced, such as de Janeiro (FAPERJ) and Conselho Nacional de different variations in temperature tolerance and Desenvolvimento Científico e Tecnológico (CNPq). other adaptive traits. In this sense, Olivera et al. RESUMO (2007) isolated bacteria of different genera with proteolytic activity in the sediment samples from Duas amostras de bactérias psicrófilas foram isoladas subantarctic areas in Argentina, characterizing do solo da Ilha do Rei Jorge, na Antártica. A análise their proteases and their thermokinesis. The results filogenética baseada no gene do RNAr 16S(rrs) showed that the thermodynamic parameters of the levou à correlação destes isolados com Sporosarcina enzymes varied considerably among the different aquimarina (99%) e Algoriphagus antarcticus (99%), isolates, which suggests important variations com porcentagem de alinhamento de 99% e 98%, in the thermo-properties of the proteases in this respectivamente. O sobrenadante do meio de cultura de environment relative to bacterial original genus. ambos os isolados apresentou atividades proteolíticas In addition, enzymes produced by psychrophilics detectadas por eletroforese em gel de poliacrilamida can offer biotechnological advantages to be contendo dodecil sulfato de sódio e gelatina como used in different areas, such as food processing, substrato proteico. Sob estas condições, S. aquimarina laundry powder for cold-water washing, leather apresentou uma protease de 55 kDa com melhor industry as well as in the treatment of effluents atividade em pH 7,0 e a 27°C. Também foi detectada and bioremediation of polluted soils and waste uma única protease extracelular em A. antarcticus, no waters in cold environment (Demain 1999, entanto com massa molecular em torno de 90 kDa e com Van den Burg 2003). Moreover, the capacity of melhor atividade em pH 9,0 e a 37°C. As proteases de these psychrophilic microorganisms, including ambos os isolados foram inibidas por 1,10-fenantrolina S. aquimarina and A. antarcticus, to grow and e EDTA, dois inibidores de metaloproteases. Este é o produce enzymes at low temperatures is beneficial primeiro registro de detecção de proteases em ambas as it fosters substantial energy saving in large scale as espécies, e nossos resultados podem contribuir para enzyme production processes, since there is no ampliar o conhecimento básico de proteases provenientes need to heat up bioreactors (Margesin and Feller do ambiente antártico e podem ajudar na prospecção de 2010). Our results demonstrate that S. aquimarina futuras aplicações biotecnológicas dessas enzimas.

An Acad Bras Cienc (2015) 87 (1) 118 ANDERSON F. SANTOS et al.

Palavras-chave: Algoriphagus antarcticus, Antártica, LOWRY OH, ROSEBOUGH NJ, FARRA L AND RANDALL RJ. 1951. Sporosarcina aquimarina, Protease. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275. MARGESIN R AND FELLER G. 2010. Biotechnological applica­ REFERENCES tions of psychrophiles. Environ Technol 31: 835-844. BERTANI G. 1951. Studies on lysogenesis. I. The mode of phage MASSOL-DEYA AA, ODELSON DA, HICKEY RF AND TIEDJE JM. liberation by lysogenic Escherichia coli. J Bacteriol 62: 1995. Bacterial community fingerprinting of amplified 16S 293-300. and 16–23S ribosomal DNA gene sequences and restriction CASANUEVA A, TUFFIN M, CARY C AND COWAN DA. 2010. endonuclease analysis (ARDRA). In: AKKERMANS AD Molecular adaptations to psychrophily: the impact of et al. (Eds), Molecular Microbial Ecology Manual, Berlin: “omic” technologies”. Trends Microbiol 18: 374-381. Springer, p. 1-8. CAVICCHIOLI R, SIDDIQUI KS, ANDREWS D AND SOWERS KR. MORITA RY. 1975. Psychrophilic bacteria. Bacteriol Rev 39: 2002. Low-temperature extremophiles and their applications. 144-167. Curr Opin Biotechnol 13: 253-261. OLIVERA NL, SEQUEIROS C AND NIEVAS ML. 2007. Diversity CAVICCHIOLI R AND THOMAS T. 2000. Extremophiles. and enzyme properties of protease-producing bacteria In: LEDERBERG J ET AL. (Eds), Encylopedia of isolated from sub-Antarctic sediments of Isla de Los Microbiology, San Diego: Academic Press, p. 317-337. Estados, Argentina. Extremophiles 11: 517-526. CHOR B, HENDY MD AND SNIR S. 2006. Maximum likelihood OLSON JJ. 2002. Antarctica: a review of recent medical research. jukes-cantor triplets: analytic solutions. Mol Biol Evol Trends Pharmacol Sci 23: 487-490. 23: 626-632. POINTING SB, CHAN Y, LACAP DC, LAU MC, JURGENS JA AND D’AVILA-LEVY CM, SANTOS ALS, CUERVO P, JESUS JB AND FARRELL RL. 2009. Highly specialized microbial diversity BRANQUINHA MH. 2012. Applications of zymography in hyper-arid polar desert. Proc Natl Acad Sci USA 106: (substrate-SDS-PAGE) for peptidase screening in a post- 19964-19969. genomic era. In: MAGDELDIN S (Ed), Gel Electrophoresis RAO MB, TANKSALE AM, GHATGE MS AND DESHPANDE VV. - Advanced Techniques, InTech 1: 265-288. 1998. Molecular and biotechnological aspects of microbial DEMAIN AL. 1999. Pharmaceutically active secondary proteases. Microbiol Mol Biol Rev 62: 597-635. metabolites of microorganisms. Appl Microbiol Biotechnol RUSSELL NJ. 1998. Molecular adaptations in psychrophilic 52: 455-463. bacteria: potential for biotechnological applications. Adv DOJKA MA, HARRIS JK AND PACE NR. 2000. Expanding the Biochem Engin Biotechnol 61: 1-21. known diversity and environmental distribution of an SIMÕES JC, ARIGONY NJ AND BREMER UF. 2004. O uso de mapas uncultured phylogenetic division of bacteria. Appl Environ antárticos em publicações. Pesq Antart Brasil 4: 191-197. Microbiol 66: 1617-1621. STACKEBRANDT E AND EBERS J. 2006. Taxonomic parameters EWING B AND GREEN P. 1998. Base-calling of automated revisited: tarnished gold standards. Microbiol Today 33: sequencer traces using phred. II. Error probabilities. Gen 153-155. Res 8: 186-194. TEIXEIRA LCRS, PEIXOTO RS, CURY JC, SUL WJ, PELLIZARI FELLER G AND GERDAY C. 2003. Psychrophilic enzymes: hot VH, TIEDJE J AND ROSADO AS. 2010. Bacterial diversity topics in cold adaptation. Nature Rev Microbiol 1: 200-208. in rhizosphere soil from Antarctic vascular plants of GASCUEL O AND STEEL M. 2006. Neighbor-Joining Revealed. Admiralty Bay, maritime Antarctica. ISME J 4: 989-1001. Mol Biol Evol 23: 1997-2000. THOMAS DN AND DIECKMANN GS. 2002. Antarctic Sea ice - a GERDAY C, AITTALEB M, ARPIGNY JL, BAISE E, CHESSA JP, habitat for extremophiles. Science 295: 641-644. GARSOUX G, PETRESCU I AND FELLER G. 1997. Psychrophilic VAN DEN BURG B. 2003. Extremophiles as a source for novel enzymes: a thermodynamic challenge. Biochim Biophys enzymes. Curr Opin Microbiol 6: 213-218. Acta 1342: 119-131. VAN TRAPPEN S, VANDECANDELAERE I, MERGAERT J AND GERDAY C ET AL. 2000. Cold-adapted enzymes: from fundamentals SWINGS J. 2004. Algoriphagus antarcticus sp. nov., a novel to biotechnology. Trends Biotechnol 18: 103-107. psychrophile from microbial mats in Antarctic lakes. Int J GRATIA E, WEEKERS F, MARGESIN R, D’AMICO S, THONART P System Evol Microbiol 54: 1969-1973. AND FELLER G. 2009. Selection of a cold-adapted bacterium VAZQUEZ SC, CORIA SH AND MAC CORMACK WP. 2004. for bioremediation of wastewater at low temperatures. Extracellular proteases from eight psychrotolerant antarctic Extremophiles 13: 763-768. strains. Microbiol Res 159: 157-166. HEUSSEN C AND DOWDLE EB. 1980. Electrophoretic analysis of VAZQUEZ SC, HERNANDEZ E AND MAC CORMACK WP. plasminogen activators in polyacrylamide gels containing 2008. Extracellular proteases from the Antarctic marine sodium dodecyl sulfate and copolymerized substrates. Anal Pseudoalteromonas sp. P96-47 strain. Rev Argent Biochem 102: 196-202. Microbiol 40: 63-71. ISHII K AND FUKUI M. 2001. Optimization of annealing temperature WYNN-WILLIAMS DD. 1996. Antarctic microbial diversity: the to reduce bias caused by a primer mismatch in multitemplate basis of polar ecosystem processes. Biodivers Conserv 5: PCR. Appl Environm Microbiol 67: 3753-3755. 1271-1129.

An Acad Bras Cienc (2015) 87 (1) PROTEASES FROM S. aquimarina AND A. antarcticus 119

YERGEAU E AND KOWALCHUK GA. 2008. Responses of Antarctic YOON JH, LEE KC, WEISS R, KHO YH, KANG KH AND soil microbial communities and associated functions to PARK YH. 2001. Sporosarcina aquimarina sp. nov., a temperature and freeze–thaw cycle frequency. Environm bacterium isolated from seawater in Korea, and transfer of Microbiol 10: 2223-2235. Bacillus globisporus (Larkin and Stokes 1967), Bacillus psychrophilus (Nakamura 1984) and Bacillus pasteurii (Chester 1898) to the genus Sporosarcina as Sporosarcina globispora comb. nov., Sporosarcina psychrophila comb. nov. and Sporosarcina pasteurii comb. nov., and emended description of the genus Sporosarcina. Int J System Evol Microbiol 51: 1079-1086.

An Acad Bras Cienc (2015) 87 (1)